Preparing mosquitoes for identification and screening

Mosquitoes collected in the field can be preserved for purposes of identification and screening to see whether or not they harbour any arboviruses. Here, we look at the steps involved in preparing both adult and larval mosquitoes.

Adult mosquitoes

The taxonomic features that are used for identification involve the morphology, or structural features of the body, and the colour and position of setae, the stiff bristles present on the body of the mosquito. Adult mosquitoes are always preserved dry to allow these features to be observed.

The traditional method of preparing adult mosquitoes is by pinning: the mosquito is killed by exposure to ethyl acetate vapour, then pinned through the thorax to a small stage (Figure 1). The stage is typically held by a larger pin that is labelled with details of the location of the collection site with GIS reference, type of habitat, date of collection, collector, and species (if known).

Figure 1: Arrangement of a pinned mosquito specimen.1

More recently, card pointing has been used. Card points are prepared by cutting Bristol board into small triangular shapes and attaching them to a large pin. A small amount of adhesive is applied to the tip of the card point, and this is lowered on to the middle of the thorax of the mosquito (Figure 2).

Figure 2: Site of adhesion of card point to the mosquito specimen | Figure 3: Arrangement of a card point mosquito specimen with labels.1

Mosquito larvae

Mosquito larvae should not be presented dry but in water soluble media, such as Berlese’s fluid, Gater’s fluid or Hoyer’s fluid. To prepare the specimen, sufficient fluid should be placed on a microscope slide and the larva lowered into it dorsal side up. A coverslip can then be applied over the specimen, and this can be sealed with a waterproof substance such as nail varnish. Finally, the slide should be labelled with details of the specimen.

Screening field mosquitoes for Zika virus

Zika virus is a member of the genus Flavivirus (family Flaviviridae), a group of viruses that use RNA as its genetic material rather than DNA, which stores genetic information in animals and plants. The virus consists of single strands of RNA enclosed in a protein coat, which is further covered in a membrane called a viral envelope. To determine if a mosquito has Zika virus it is necessary to extract this RNA from the mosquito.

RNA can degrade rapidly at room temperature, so mosquitoes collected from the field must be stored appropriately as soon as possible. This can be by using cold temperatures (-80°C freezers, liquid nitrogen and dry ice) or commercially available reagents that preserve RNA. Stored mosquitoes can then be processed in the laboratory.

Figure 3 outlines the steps from extraction of RNA to replication, making it possible to detect small quantities of viral genetic material in a wild caught mosquito. In addition to being very sensitive this process is very specific, and can be designed to detect Zika but not other viruses that the mosquito may carry.

Figure 3. Process of preparing and amplifying Zika genetic material to allow detection of the virus in wild caught mosquitoes. cDNA: synthetic form of DNA that is made using RNA as a template. PCR: polymerase chain reaction, a technique used to generate many copies of a specific stretch of DNA or cDNA from a small sample.

An example of the methodology to detect Zika virus in field-caught mosquitoes is described by Faye and colleagues2 in which Zika virus was detected in 31 of 1969 mosquito pools from Aedes and Mansonia mosquitoes collected in Southeastern Senegal.